U.S. patent application number 13/667775 was filed with the patent office on 2014-05-08 for synthetically functionalized living cells.
This patent application is currently assigned to Massachusetts Institute of Technology. The applicant listed for this patent is MASSACHUSETTS INSTITUTE OF TECHNOLOGY. Invention is credited to Robert E. Cohen, Darrell J. Irvine, Michael F. Rubner, Albert J. Swiston.
Application Number | 20140127774 13/667775 |
Document ID | / |
Family ID | 50622714 |
Filed Date | 2014-05-08 |
United States Patent
Application |
20140127774 |
Kind Code |
A1 |
Swiston; Albert J. ; et
al. |
May 8, 2014 |
Synthetically Functionalized Living Cells
Abstract
Uniform, functional polymer patches can be attached to a
fraction of the surface area of living individual cells. These
surface-modified cells remain viable after attachment of the
functional patch. The patch does not completely occlude the
cellular surface from the surrounding environment. Functional
payloads carried by the patch may include, for example, drugs or
other small molecules, peptides, proteins, thermally responsive
polymers, and nanoparticles, or any other material that can be
incorporated in a polymer patch of subcellular dimensions. The
patch can include one or more polyelectrolyte multilayers
(PEMs).
Inventors: |
Swiston; Albert J.;
(Baltimore, MD) ; Rubner; Michael F.; (Westford,
MA) ; Cohen; Robert E.; (Jamaica Plain, MA) ;
Irvine; Darrell J.; (Arlington, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
MASSACHUSETTS INSTITUTE OF TECHNOLOGY |
Cambridge |
MA |
US |
|
|
Assignee: |
Massachusetts Institute of
Technology
Cambridge
MA
|
Family ID: |
50622714 |
Appl. No.: |
13/667775 |
Filed: |
November 2, 2012 |
Current U.S.
Class: |
435/178 ;
435/180 |
Current CPC
Class: |
C12N 2533/30 20130101;
C12N 11/08 20130101; C12N 5/0068 20130101; C12N 2533/80
20130101 |
Class at
Publication: |
435/178 ;
435/180 |
International
Class: |
C12N 11/08 20060101
C12N011/08 |
Goverment Interests
FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This invention was made with government support under
contract number DMR 0213282 awarded by the National Science
Foundation. The government has certain rights in the invention.
Claims
1. A composition comprising: a cell; and a polymer patch associated
with a fractional portion of the cell surface; wherein the polymer
patch includes: a cytophilic face having a specific affinity for
the cell surface and being substantially in contact with the cell
surface; and an exposed face.
2. The composition of claim 1, wherein the polymer patch further
includes a functional layer intermediate to the cytophilic face and
the exposed face.
3. The composition of claim 2, wherein the functional layer
includes a fluorescent material, a magnetic material, or a
drug.
4. The composition of claim 1, wherein the cytophilic face includes
hyaluronic acid, chitosan, biotin, or an adhesive peptide.
5. The composition of claim 1, wherein the cytophilic face includes
a polyelectrolyte multilayer.
6. The composition of claim 1, wherein the exposed face includes a
polyelectrolyte multilayer.
7. The composition of claim 2, wherein the functional layer
includes a polyelectrolyte multilayer.
8. The composition of claim 1, wherein the polymer patch has
lateral dimensions in the range of 1 .mu.m to 250 .mu.m and a
thickness in the range of 50 nm to 1 .mu.m.
9-35. (canceled)
36. A method of attaching a layer to a surface of a cell,
comprising: exposing a cell to a polymer structure having a
cytophilic layer; and releasing a portion of the polymer structure
to expose the layer.
Description
CLAIM OF PRIORITY
[0001] This application claims priority to provisional U.S. Patent
Application No. 61/043,592, filed Apr. 9, 2008, which is
incorporated by reference in its entirety.
TECHNICAL FIELD
[0003] This invention relates to synthetically functionalized
living cells.
BACKGROUND
[0004] Polyelectrolyte multilayers can be easily assembled on a
variety of surfaces. Selection of the materials, assembly
conditions, and post-processing conditions can be used to control
the chemical, biological, structural and optical properties of the
final product. Polyelectrolyte multilayers have previously been
used in several biological applications, including drug delivery,
biomaterial coatings, and precisely functionalizing surfaces to
control adherent cellular growth.
SUMMARY
[0005] Polymer discs or bodies, or "patches," can be deposited onto
the surface of a living cell, and this patch may serve as a broad
platform to affect cellular behavior and functionality. These
patches can be used to confer non-native functions and
characteristics (including, for example, chemical, enzymatic,
fluorescent, magnetic, or other characteristics) to the cell, while
allowing the cell to perform its natural functions as well, since
the patch covers only a portion of the cell surface.
[0006] A large number of patches can be fabricated over a large
area (e.g., approximately 1 in.sup.2 or larger) of a substrate
simultaneously (using, e.g., lithographic techniques, though
fabrication is not limited to lithography). Patches can be readily
prepared using techniques for forming polyelectrolyte multilayers
(PEMs). As fabricated on the substrate, each patch includes a
labile layer that can dissociate under precisely selected
conditions to release the layer(s) above (for example, which can
include layers having affinity for cells and selected "payload"
layers, which can include materials such as drugs or nanoparticles)
from the substrate. Advantageously, the labile layer can undergo
dissociation in the same solution (pH, salt concentration, etc.)
and temperature conditions used to contact the cells to the
patches. Thus, patch attachment to cells and patch dissociation
from the substrate can occur in a single step.
[0007] In one aspect, a composition includes a cell, and a polymer
patch associated with a fractional portion of the cell surface,
where the polymer patch includes a cytophilic face having a
specific affinity for the cell surface and being substantially in
contact with the cell surface, and an exposed face.
[0008] The polymer patch can further include a functional layer
intermediate to the cytophilic face and the exposed face. The
functional layer can include a fluorescent material, a magnetic
material, or a drug. The cytophilic face can include hyaluronic
acid, chitosan, biotin, or an adhesive peptide. The cytophilic face
can include a polyelectrolyte multilayer. The exposed face can
include a polyelectrolyte multilayer. The polymer patch can have
lateral dimensions in the range of 1 .mu.m to 250 .mu.m and a
thickness in the range of 50 nm to 1 .mu.m.
[0009] In another aspect, a polymer structure arranged on a
substrate includes a substrate-adhering layer, a labile layer
configured to selectively dissociate under predetermined conditions
arranged over the substrate-adhering layer, and a cytophilic layer
arranged over the labile layer, the cytophilic layer having a
specific affinity for a surface of a predetermined cell type, where
the cytophilic layer has a surface area smaller than a cell of the
predetermined cell type.
[0010] The structure can include a diffusion barrier layer arranged
intermediate to the labile layer and the cytophilic layer. The
structure can include a functional layer arranged intermediate to
the labile layer and the cytophilic layer. The substrate-adhering
layer can include a polyelectrolyte multilayer. The
substrate-adhering layer can include a polyelectrolyte multilayer.
The labile layer can include a hydrogen-bonded polymer multilayer.
The cytophilic layer can include a polyelectrolyte multilayer.
[0011] In another aspect, a polymer structure arranged on a
substrate includes a substrate-adhering layer including a
polyelectrolyte multilayer, a labile layer including a
hydrogen-bonded polymer multilayer arranged over the
substrate-adhering layer, and a cytophilic layer including a
polyelectrolyte multilayer arranged over the labile layer, the
cytophilic layer having a specific affinity for a surface of a
predetermined cell type, and where the cytophilic layer has a
surface area smaller than a cell of the predetermined cell
type.
[0012] The structure can include a diffusion barrier layer
including a polyelectrolyte multilayer arranged intermediate to the
labile layer and the cytophilic layer; or a functional layer
including a polyelectrolyte multilayer arranged intermediate to the
labile layer and the cytophilic layer
[0013] The cytophilic layer can include a ligand for a cell surface
receptor of the predetermined cell type. The labile layer can be
configured to dissolve under conditions conducive to binding of the
cell to the cytophilic layer. The structure can be a member of a
population of substantially identical polymer structures arranged
on a substrate.
[0014] The structure can have lateral dimensions in the range of 1
.mu.m to 250 .mu.m and a thickness in the range of 50 nm to 1
.mu.m. The structure can have lateral dimensions in the range of 1
.mu.m to 100 .mu.m. The structure can have lateral dimensions in
the range of 1 .mu.m to 50 .mu.m.
[0015] In another aspect, a method of making a composition includes
forming a polymer structure arranged on a substrate, the polymer
structure including: a substrate-adhering layer including a
polyelectrolyte multilayer, a labile layer including a
hydrogen-bonded polymer multilayer arranged over the
substrate-adhering layer, and a cytophilic layer including a
polyelectrolyte multilayer arranged over the labile layer, the
cytophilic layer having a specific affinity for a surface of a
predetermined cell type, where the cytophilic layer has a surface
area smaller than a cell of the predetermined cell type, and
contacting the polymer structure with a cell of the predetermined
cell type, thereby forming a cell-patch-substrate association, and
causing the labile layer to release a cell-patch association from
the substrate.
[0016] Contacting the polymer structure with a cell of the
predetermined cell type can occur under conditions in which the
labile layer is substantially soluble. The polymer structure can
further include a diffusion barrier layer including a
polyelectrolyte multilayer arranged intermediate to the labile
layer and the cytophilic layer. The polymer structure can further
include a functional layer including a polyelectrolyte multilayer
arranged intermediate to the labile layer and the cytophilic layer.
The cytophilic layer includes a ligand for a cell surface receptor
of the predetermined cell type. The structure can be a member of a
population of substantially identical polymer structures arranged
on a substrate.
[0017] Causing the labile layer to release a cell-patch association
from the substrate can include dissociating the labile layer.
Dissociation can include, for example, dissolving hydrogen-bonded
films, mechanical stress, and temperature induced dissolution, or a
combination thereof. Contacting the polymer structure with a cell
of the predetermined cell type can occur before, simultaneously
with, or after causing the labile layer to release a cell-patch
association from the substrate.
[0018] In another aspect, a method of attaching a layer to a
surface of a cell includes exposing a cell to a polymer structure
having a cytophilic layer, and releasing a portion of the polymer
structure to expose the layer.
[0019] The details of one or more embodiments are set forth in the
accompanying drawings and the description below. Other features,
objects, and advantages will be apparent from the description and
drawings, and from the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] FIGS. 1A-1C are schematic depictions of patches and their
associations with cells. FIG. 1D is an overview of the cell
functionalization scheme. Panel (a) shows a regular array of
surface-bound patches spaced 50 .mu.m apart. The green fluorescence
is from FITC-PAH. Panel (b) shows that after cell incubation and
attachment, a majority of the surface-bound patches are occupied.
Panel (c) shows that the patches were released from the surface
while remaining attached to the cell membrane.
[0021] FIG. 2 is a schematic diagram of a method of making
patches.
[0022] FIG. 3 is a schematic diagram of a method of making
patches.
[0023] FIG. 4 is a fluorescence micrograph of patches.
[0024] FIG. 5 is a fluorescence micrograph of patches.
[0025] FIG. 6 is a fluorescence micrograph of beads interacting
with patches.
[0026] FIGS. 7A-7B are fluorescence micrographs of patches.
[0027] FIGS. 8A-8B are fluorescence micrographs of patches.
[0028] FIG. 9 is a micrograph showing cells attached to
patches.
[0029] FIG. 10 is a micrograph showing cells attached to
patches.
[0030] FIGS. 11A-11B are micrographs of cells attached to
patches.
[0031] FIG. 12 is a graph depicting patch dissolution behavior.
[0032] FIG. 13 is a graph depicting patch dissolution behavior.
[0033] FIG. 14 is a graph depicting patch dissolution behavior.
[0034] FIG. 15 is a fluorescence micrograph of patches.
[0035] FIG. 16 is a graph depicting patch-cell interaction
behavior.
[0036] FIGS. 17A-17B are micrographs depicting patch-cell
interaction behavior.
[0037] FIG. 18 is a group of images depicting a fluorescent patch
associated with a single cell.
[0038] FIG. 19 is a time sequence of images depicting a magnetic
patch associated with an individual cell.
[0039] FIG. 20 is graph depicting thermal characteristics of a
patch.
[0040] FIGS. 21A-21C are micrographs depicting patches.
[0041] FIGS. 22A-22C are a group of images depicting a fluorescent
patch associated with a single cell.
[0042] FIGS. 23A-23C are a group of images depicting a fluorescent
patch associated with a single cell.
[0043] FIG. 24 is a graph depicting optical properties of
patches.
[0044] FIGS. 25A-25C are a group of images depicting a fluorescent
patch associated with a single cell.
[0045] FIGS. 26A-26C are a group of images depicting a fluorescent
patch associated with a single cell.
[0046] FIG. 27 is a time sequence of images depicting a magnetic
patch associated with an individual cell.
DETAILED DESCRIPTION
[0047] Synthetic materials have been interfaced with biological
systems for cellular encapsulation applications. Since at least
1964, encapsulation strategies for cellular cargoes have focused on
wrapping a cell or cell aggregate in a protective polymer shell to
prevent an autoimmune reaction from deactivating the payload, while
allowing small species through the encapsulating semipermeable
membrane (see, e.g., Thomas M. S. Chang, Science 146 (3643), 524
(1964), which is incorporated by reference in its entirety). Many
cell-based therapies, such as those that encapsulate islets cells
for diabetes therapy, prefer this immunoisolation approach to
systemic immunosupression. See, for example, Gorka Orive, et al.,
Nat Med 9 (1), 104 (2003); Hasan Uludag, et al., Advanced Drug
Delivery Reviews 42 (1-2), 29 (2000); and Surita R. Bhatia, et al.,
Current Opinion in Colloid & Interface Science 10 (1-2), 45
(2005), each of which is incorporated by reference in its
entirety.
[0048] Research on polyelectrolyte multilayer encapsulation has
primarily focused on uniformly coating the surface of approximately
spherical bodies, e.g., living or fixed cells. However, the
approaches used restricted the accessibility of the cellular
surface to the environment. An approach by which functionality is
introduced to a relatively small fraction of a cellular surface
(i.e., a "patch") can allow the majority of the cellular surface to
remain free to interact with the environment.
[0049] Cellular encapsulation strategies have typically completely
occluded the surface of the cell from direct contact with its
environment. Past efforts have included using polymer multilayer
assemblies on the surface of living and dead, fixed cells. See, for
example, Matthieu Germain, et al., Biosensors and Bioelectronics 21
(8), 1566 (2006); A. Diaspro, et al., Langmuir 18 (13), 5047
(2002); S. Krol, et al., Langmuir 21 (2), 705 (2005); A. L.
Hillberg and M. Tabrizian, Biomacromolecules 7 (10), 2742 (2006);
S. Moya, et al., Colloids and Surfaces A: Physicochemical and
Engineering Aspects 183-185, 27 (2001); and R. Georgieva, et al.,
Langmuir 20 (5), 1895 (2004), each of which is incorporated by
reference in its entirety.
[0050] For cells that elute a species of interest (for example, a
metabolite) that can diffuse through a protective membrane,
encapsulation may be acceptable. However, for cells that require
direct environmental contact to perform their desired functions,
these encapsulation strategies can limit the usefulness of such
cells. For example, the burgeoning fields of immune system
engineering, adoptive T-cell therapies, and advanced cell-based
vaccines realize little benefit from the traditional encapsulation
paradigm.
[0051] Cellular passivation is only one possible option for useful
cellular functionalization. For instance, by covering only a
portion of a cellular membrane with a functional "patch," cell
surface receptors on the rest of the cell membrane can remain free
to interact with the environment. For example,
poly(diacetylene)-conjugated liposomes can be attached to the cell
surface, but many randomly attached nanometer sized "patches" were
formed. See, for example, Sofiya Kolusheva, et al., Angewandte
Chemie International Edition 44 (7), 1092 (2005), which is
incorporated by reference in its entirety.
[0052] Uniform, multi-functional polymer patches can be attached to
cells, and the patches subsequently released from an underlying
substrate when exposed to physiological pH conditions and
cell-compatible temperatures (e.g., between 4.degree. C. and
37.degree. C.). These engineered heterostructures can include both
a "payload" component (such as, for example, superparamagnetic
nanoparticles) and a cell-adhesive face that includes a component
that has an affinity for the cells in question. For example, in the
case of lymphocytes, the cell-adhesive face can include hyaluronic
acid. For other cell types, cell-type specific antibodies can be
employed to attach the patch to the membrane. The cells can attach
to the patch before the patch releases from the surface, thus
yielding a synthetically functionalized and living cell. The
patches are also referred to as "backpacks," as they share certain
characteristics with ordinary backpacks, such as portability, the
ability to carry any desired cargo, and can be borne without
impairing the functioning of the wearer.
[0053] One method to create the desired patch is with a
polyelectrolyte multilayer. In some circumstances, polyelectrolyte
multilayers can also confer desirable optical properties to
surfaces, such as anti-reflectivity, or reflectivity in a desired
range of wavelengths (see, for example, U.S. Patent Application
Publication Nos. 2003/0215626 and 2006/0029634), and/or desirable
surface energy characteristics. See, for example, U.S. Patent
Application Publication Nos. 2006/0029634, which is incorporated by
reference in its entirety.
[0054] A polyelectrolyte has a backbone with a plurality of charged
functional groups attached to the backbone. A polyelectrolyte can
be polycationic or polyanionic. A polycation has a backbone with a
plurality of positively charged functional groups attached to the
backbone, for example poly(allylamine hydrochloride) (PAH). A
polyanion has a backbone with a plurality of negatively charged
functional groups attached to the backbone, such as sulfonated
polystyrene (SPS) or poly(acrylic acid) (PAA), or a salt thereof.
Some polyelectrolytes can lose their charge (i.e., become
electrically neutral) depending on conditions such as pH. Some
polyelectrolytes, such as copolymers, can include both polycationic
segments and polyanionic segments.
[0055] Layer-by-layer processing of polyelectrolyte multilayers can
be used to make conformal thin film coatings with molecular level
control over film thickness and chemistry. Charged polyelectrolytes
can be assembled in a layer-by-layer fashion. In other words,
positively- and negatively-charged polyelectrolytes can be
alternately deposited on a substrate. One method of depositing the
polyelectrolytes is to contact the substrate with an aqueous
solution of polyelectrolyte at an appropriate pH. The pH can be
chosen such that the polyelectrolyte is partially or weakly
charged, or such that the polyelectrolyte is substantially
completely or strongly charged. The multilayer can be described by
the number of bilayers it includes, a bilayer resulting from the
sequential application of oppositely charged polyelectrolytes. For
example, a multilayer having the sequence of layers
PAH-PAA-PAH-PAA-PAH-PAA would be said to be made of three bilayers.
Each cycle of complimentary polymers produces a complexed,
interpenetrated structure referred to as a "bilayer" and the
following notation is commonly used:
(Poly.sub.1X/Poly.sub.2Y).sub.n. Here, Poly.sub.1 and Poly.sub.2
refer to the abbreviation for the specific polymers or
nanoparticles used in a selected assembly process, X and Y refer to
the pH of the solution, and n is the number of bilayers that have
been deposited. In some cases, n may be expressed in a decimal
notation, such as for example, 3.5, which would indicate that three
bilayers of Poly.sub.1X/Poly.sub.2Y were deposited before a final
"half-bilayer" of Poly.sub.1X was deposited. In some circumstances,
charged nanoparticles can be used to as one or both of the charged
species. See, for example, Lee, D., et al., Nano Letters, 6, 2305
(2006), which is incorporated by reference in its entirety.
[0056] The properties of weakly charged polyelectrolytes can be
precisely controlled by changes in pH. See, for example, G. Decher,
Science 1997, 277, 1232; Mendelsohn et al., Langmuir 2000, 16,
5017; Fery et al., Langmuir 2001, 17, 3779; Shiratori et al.,
Macromolecules 2000, 33, 4213; and U.S. patent application Ser. No.
10/393,360, each of which is incorporated by reference in its
entirety. A coating of this type can be applied to any surface
amenable to the water based layer-by-layer (LbL) adsorption process
used to construct these polyelectrolyte multilayers. Because the
water based process can deposit polyelectrolytes wherever the
aqueous solution contacts a surface, even the inside surfaces of
objects having a complex topology can be coated. In general, a
polyelectrolyte can be applied to a surface by any method amenable
to applying an aqueous solution to a surface, such as dipping or
spraying.
[0057] Other modifications of a deposited polyelectrolyte
multilayer are possible. For example, a nonporous polyelectrolyte
multilayer can form porous thin film structures induced by a simple
acidic, aqueous process. Tuning of this pore forming process, for
example, by the manipulation of such parameters as salt content
(ionic strength), temperature, or surfactant chemistry, can lead to
the creation of micropores, nanopores, or a combination thereof. A
nanopore has a diameter of less than 150 nm, for example, between 1
and 120 nm or between 10 and 100 nm. A nanopore can have diameter
of less than 100 nm. A micropore has a diameter of greater than 150
nm, typically greater than 200 nm. Selection of pore forming
conditions can provide control over the porosity of the coating.
For example, the coating can be a nanoporous coating, substantially
free of micropores. Alternatively, the coating can be a microporous
coating having an average pore diameters of greater than 200 nm,
such as 250 nm, 500 nm, 1 micron, 2 microns, 5 microns, 10 microns,
or larger.
[0058] Desired chemistries can be included in the polyelectrolyte
multilayers. The chemistry can be added during manufacture of the
multilayer, or after manufacture. For example, antibacterial
chemistries (such as silver nanoparticles or quaternary ammonium
salts) can be included in the multilayer during manufacture. The
resulting multilayer can then have desired properties (such as
antibacterial properties) arising from the incorporated chemistry.
In some circumstances, the chemistry can be controllably released
from the multilayer.
[0059] A patch can be fabricated and functionalized ex vivo. In
other words, the patch can be prepared in an environment
substantially free of cells. An ex vivo approach can offer more
opportunities in geometry, functionalization chemistries, and
solvent conditions than does in vivo fabrication.
[0060] In general, a patch can have a multilayer structure.
Typically the patch is fabricated on a substrate before being
introduced to cells. The substrate or patch can optionally include
an adhesion layer, depending on the substrate, selected to provide
desired mechanical robustness to the patch on the substrate. The
next layer is a labile releasable layer. The labile layer is
composed of non-cytotoxic polymers. The labile layer can include
polymers that dissociate in neutral pH conditions, such as H-bonded
multilayer films, biopolymers digested by specific enzymes (such as
cellulase digestion of cellulose derivatives), hydrolysable
polymers, or polymer systems with temperature-dependant solubility
(lower critical solution temperature, "LCST", behavior in
particular). H-bonded multilayer films can include polymers such as
poly(acrylic acid) (PAA), poly(methylacrylic acid) (PMAA),
poly(ethylene glycol) (PEG), poly(vinylpyrrolidone) (PVPON),
poly(N-vinylcaprolactam) (PVCL), and poly(N-isopropylacrylamide)
(PNIPAAm). The films can include but are not limited to PAA/PEG,
PMAA/PEG, PMAA/PVPON, PMAA/PVCL, or PMAA/PNIPAAm films. In general,
the labile layer is selectively removable, under controllable
conditions, such that the remaining layers of the patch (described
below) are released from the substrate.
[0061] The patch can include an optional diffusion barrier,
depending on the diffusion characteristics of the functional layer
(if present) and of the cytophilic layer. The cytophilic layer
includes at least one polymer or other component that has a strong
affinity to attach to a cell surface. For example, the cytophilic
layer can include extracellular matrix (ECM) polymers (such as
hyaluronic acid), or polymers functionalized with adhesive
moieties, such as biotin, or adhesive peptides, such as antibodies
or RGD-containing peptides. The materials and properties of the
cytophilic layer can be chosen to favor attachment of the patch to
a desired type of cell. For example, antibodies present in the
cytophilic layer can promote attachment of cells bearing the
corresponding antigen over cells that do not present the antigen.
The cytophilic layer can include reactive groups capable of forming
covalent bonds with functional groups on the cell surface.
Cell-patch interactions are further discussed below.
[0062] A multilayer patch can be fabricated on a planar surface.
Next, cells are introduced, and attach to the outermost cytophilic
layer. Last, the labile layer is caused to dissolve or dissociate,
an event that can be tuned depending on the nature of the diffusion
barrier layer (such as thickness and pH assembly conditions) and
environmental conditions such as salinity and temperature. The
cells are thus freed from the surface, departing with a polymeric
patch attached to its surface. In other words, the cell is
partially encapsulated by the patch--only a fraction of the cell
surface is covered by the patch. A schematic of this approach is
illustrated in FIGS. 1A-1C.
[0063] In a modified approach, the patch can be fabricated on a
planar surface. The labile layer is next caused to dissolve or
dissociate, allowing the patches to float freely. In this free
floating state, the patches can be exposed to and become attached
to cells.
[0064] FIGS. 1A-1D illustrate the fabrication of a patch on a
substrate, its interaction with a cell, and release of the patch
and associated cell from the substrate. Arranged on substrate 110
are three patches, each including an adhesion layer 120, labile
layer 125, optional diffusion barrier 130, optional functional
layer 140, and cytophilic layer 150. In FIG. 1B, cells 160 are
exposed to the patches and individual cells become associated with
individual patches via cytophilic layers 150. FIG. 1C illustrates a
released cell following dissolution of the labile layer 125.
Partially encapsulated cell 160 includes a portion of its surface
associated with cytophilic layer 150, optional functional layer
140, and diffusion barrier 130. FIG. 1D presents another overview
of cell functionalization with patches.
[0065] One method of forming patches is to prepare the desired
multilayer assembly on a patterned "stamp," and then transfer this
through a process known as polymer-on-polymer stamping, or POPS.
POPS is an extension of microcontact printing, which requires the
fabrication of a poly(dimethylsiloxane) (PDMS) stamp on which an
"ink" is deposited. Inks used in microcontact printing have
included small molecules, such as alkanethiols and alkylsiloxanes,
as well as higher molecular weight polymers such as PAH. See, for
example, Y. Xia, and G. M. Whitesides, J. Am. Chem. Soc. 117, 3274
(1995); Y. Xia, et al., J. Am. Chem. Soc. 117, 9576 (1995); and M.
C. Berg, et al., Langmuir 19, 2231 (2003), each of which is
incorporated by reference in its entirety.
[0066] POPS uses an ink that is an assembled PEM--that is, a PEM is
built on the patterned PDMS stamp (see, e.g., P. T. H. J. Park,
Advanced Materials 16, 520 (2004), which is incorporated by
reference in its entirety). The stamp is brought into contact with
a substrate selected for its ability to interact with the ink. For
example, a gold substrate can be used when microcontact printing a
thiol-based ink; or a planar substrate coated with another charged
polymer in POPS. Specific interactions between the molecules on the
PDMS and the substrate will encourage the liftoff of the ink in a
complimentary pattern to the relief pattern on the PDMS stamp.
[0067] The POPS procedure can be sensitive to aspect ratios of the
film thickness and feature diameter. For instance, a film 5 nm
thick can easily be stamped with features of approximately 1 .mu.m
in size, but a film with a thickness of 50 nm can transfer more
effectively if the features are approximately 10 .mu.m in size.
[0068] Another method of patch fabrication is a lithographic
"lift-off" method. This method works with silica nanoparticle
assemblies and some biopolymer systems. See, for example, F. Hua,
et al., Langmuir 18, 6712 (2002); J. ShaikhMohammed, et al.,
Biomacromolecules 5, 1745 (2004); and J. ShaikhMohammed, et al.,
Langmuir 22, 2738 (2006), which is incorporated by reference in its
entirety. FIG. 2 illustrates the method schematically. First, a
substrate is uniformly coated with a positive photoresist (such as,
for example Rohm&Haas S1813). A patterned photomask (e.g., Cr
on glass) is placed above the photoresist, and the photoresist is
UV cured in the pattern of the mask. For patch fabrication, the
selected pattern can be, for example, 1-20 .mu.m wells in the
photoresist layer, although any pattern compatible with the
photolithographic method is possible. Next, a multilayer is
deposited atop of the patterned resist with the following exemplary
composition: (adhesion layer).sub.5 (labile layer).sub.x (PEM
functional layer)(cytophilic layer).sub.y. The last lithographic
step is to dissolve the photoresist in an organic solvent such as
N-methylpyrrolidone (NMP) or acetone, leaving only the polymer
posts that adsorbed directly to the substrate (see, e.g., FIG. 2).
The use of sonication during the photoresist dissolution step can
be important for desired fabrication, due to the continuity of the
film over the photoresist islands. Finally, cells can incubated
atop the posts, and attach to the cytophilic layer. Next,
dissociation of the labile layer is triggered, either by
appropriate pH or temperature conditions.
[0069] Deposition of the cytophilic biofilm directly over the
labile layer can in some circumstances render the labile layer
insoluble in phosphate buffered saline (PBS) (and thus unable to
undergo the desired dissolution). The following film composition,
including Poly(acrylamide) (PAAm), PAA, hyaluronic acid (HA), and
chitosan (CHI), was considered:
(PAAm/PAA).sub.10.5(HA/CHI).sub.3.5. The PAAm/PAA layer has been
observed to dissolve almost instantly when exposed to Milli-Q water
(pH approximately 5.5), but was insoluble (in Milli-Q or PBS) when
the HA/CHI layer was deposited on top. This may be due to the
electrostatic and hydrogen-bonding capabilities (due to numerous
hydroxyl groups along the polysaccharide) of hyaluronic acid and
chitosan. See, e.g., L. Richert et al., Langmuir 20, 448 (2004),
which is incorporated by reference in its entirety. Diffusion of
the hyaluronic acid and chitosan into the H-bonded layer may
crosslink and stabilize the release layer.
[0070] A PEM film that diffuses to only a small extent (i.e., a
diffusion barrier layer) including fully charged polyelectrolytes
can inhibit this possible diffusion process. Lavalle and co-workers
systematically probed the diffusion inhibiting behavior of
(PAH/SPS) layers within (HA/poly(L-lysine)) films (see, e.g., J. M.
Garza et al., Langmuir 20, 7298 (2004), which is incorporated by
reference in its entirety). As few as 2 bilayers of PAH/SPS were
sufficient to completely eliminate the diffusion of poly(lysine)
between multilayer compartments (see, for example, F. Boulmedais,
et al., Langmuir 19, 9873 (2003), which is incorporated by
reference in its entirety). For example, a (PAH3/SPS3) (two
fully-charged polymers under the pH conditions used) layer between
the cytophilic layer and the releasable H-bond layer, can be used
as a diffusion barrier layer. The relevant feature of the diffusion
barrier layer is that the polyelectrolytes in the layer are
substantially fully charged.
[0071] A third method of fabricating patches is a lithographic
plasma-etching method. FIG. 3 illustrates this method
schematically. A 4-ply composite stack of multilayers on a planar
substrate is first prepared. The first layer is an adhesion layer,
which encourages strong adhesion and uniformity of subsequent
stacks. The second stack is a labile layer, which will readily
dissolve in neutral or buffered conditions (see, e.g., S. A.
Sukhishvili, S. Granick, Macromolecules 35, 301 (2002); and J. Cho,
F. Caruso, Macromolecules 36, 2845 (2003), each of which is
incorporated by reference in its entirety). The cytophilic layer is
third, followed by a final labile layer. One exemplary structure
is:
(PAH4/SPS4).sub.5.5(PAA3/PAAm3).sub.20(HA3/PAH3).sub.x.5(PAAm3/PAA3).-
sub.20.
[0072] The next step is to physically mask the multilayer with a
porous structure that contains holes on the order of, for example,
1-20 .mu.m, such as developed photoresist. Next, electron-beam or
thermally evaporated gold or SiO.sub.2 can be deposited onto the
surface of the outer labile layer, and form islands 1-20 .mu.m in
diameter. These metal islands act as a physical mask for the next
step, an oxygen plasma etch (150 mTorr, 10 min) of the exposed
film. Following the plasma etching step, all that remains are 4-ply
posts of polymer multilayers, capped with a gold or SiO.sub.2
island. The last step is to dissolve the releasable (PAAm/PAA)
stacks by submerging in netural pH water, sedimenting out the
inorganic islands, and leaving the cytophilic layer suspended in
solution.
[0073] The oxygen plasma method is an isotropic etch technique.
Lateral removal (parallel with substrate surface) can occur beneath
the metal masking. However, considering the aspect ratio of the
mask to the thickness of the composite multilayer film, such
lateral removal is unlikely to be significant. Data for the
thickness of an exemplary system is shown in Table 1. If a 3 .mu.m
diameter metal island is deposited, the aspect ratio
(diameter/height) is approximately 30. Even if the rate of lateral
etching is equal to vertical removal, the size (i.e., diameter) of
the final patch will not be significantly affected.
TABLE-US-00001 TABLE 1 (PAH4/SPS4)5.5 12 nm (PAA/PAAm-800k MW)10 63
nm (HA3/FITC-PAH3)3.5 23 nm Total thickness 98 nm
[0074] The fabrication methods discussed above can provide a
polymer patch attached to a cellular surface, with only one face of
the patch exposed to solution. Each side of the patch can present
different chemical moities, resulting in a bifunctional polymer
disc. For example, biotin can be incorporated into the strong PEM
diffusion barrier during film growth, leaving the other face free
to bind to CD44 receptors on the cell. Then, a myriad of
streptavidin-conjugated molecules can be easily tagged to the cell
in very limited and highly controlled domain sizes. See, e.g., J.
M. Garza et al., Langmuir 20, 7298 (2004); and F. Boulmedais, et
al., Langmuir 19, 9873 (2003), each of which is incorporated by
reference in its entirety.
[0075] Another example of surface functionalization is to provide
non-native abilities to the cells by virtue of the associated
patches. In one example, during PEM fabrication, magnetic
nanoparticles can be impregnated into the layers, and once the
final polymer patch is attached to the surface of the cell, the
cells responded to an applied magnetic field. Any number of
functional materials, including (but not limited to) magnetic and
environmentally-responsive materials, drugs, or imaging contrast
agents, can be included in a patch. The corresponding function can
thus be provided to cells.
[0076] An in vivo method can functionalize the surface of a living
cell. A PEM film is stamped directly onto the cell surface without
first fabricating a patch as described above. This can be
accomplished by using a modified POPS protocol, similar to what has
been used to fabricate Janus-style capsules (see, e.g., Z. Li,
Macromolecules 38, 7876 (2005), which is incorporated by reference
in its entirety. A cytophilic, HA-terminated PEM can be assembled
on a patterned or unpatterned PDMS stamp. This PDMS stamp is then
lowered onto a monolayer of adherent cells, either densely packed
or ordered into a 2D periodic structure (see, e.g., H. Kim,
Advanced Functional Materials 16, 1313 (2006), which is
incorporated by reference in its entirety).
[0077] After PEM deposition onto the cell surface, well-established
chemistries, such as EDC/NHS (1-ethyl-3-(3-dimethylaminopropyl)
carbodiimide hydrochloride/N-hydroxy-succinimide), can be used to
functionalize the PEM film in addition to any functionalization
added during the PEM deposition process (that is, during PEM film
fabrication).
Non-Polyelectrolyte or Hydrogen-Bonded Multilayer Based Patch
Fabrication Techniques
[0078] Other methods can fabricate a patch that will perform
similarly or identically to patches made using layer-by-layer
techniques. For instance, a heterostructured patch may be made by
"inking" (e.g., non-selectively adsorbing to the surface) several
regions of polymer onto a patterned PDMS stamp, and then
transferring this pattern using polymer-on-polymer (POPS)
techniques. Similarly, polymer layers can be applied from solutions
using spin coating, dip coating, spray coating, surface grafting,
vapor deposition, fluidized bed coating, roller coating, or
meniscus coating, and may be patterned using a PDMS stamp with POPs
techniques, a lithographically patterned substrate with selective
liftoff, or a patterned barrier or mask. A key design criteria for
any solution-based deposition technique is that the solvent system
used to apply a given layer onto a previously applied layer must
not substantially dissolve or disrupt the previous layer. For
instance, a labile region of PNIPAAm homopolymer or other material
with a chemically, thermally or mechanically triggered release
capability may be applied to a surface from a water based solution,
and a polymer functionalized with cell interacting moieties such as
adhesion tripeptides like RGD can be applied to this previous layer
from non-aqueous solutions. Suitable non-water soluble polymers
include functionalized polyamides, polyesters, polyurethanes,
acrylic copolymers, and methacrylic copolymers, and polysaccharides
such as chitin, and cellulose, to name a few. Suitable
water-soluble polymer solutions include PAA, PMAA, PVPON, PAH,
poly(vinyl pyridine), poly(vinyl alcohol), PEG, PAAm, PDAC, SPS,
and water soluble polysaccharides such as chitosan,
carboxymethylcellulose, hyaluronic acid, dextran, and alginate, to
name a few. Synthetic and natural derivatives of both the non-water
soluble and water-soluble polymers may also be considered.
EXAMPLES
[0079] Bifunctional patches can be fabricated using a POPS
technique. First, a PDMS stamp was made from a Si master (e.g., a
Si wafer covered in a patterned SU-8 photoresist layer), and a
composite polyelectrolyte multilayer/hydrogen-bonded multilayer
stack built on top of it. This composite structure can generally
include at least four distinct layers. The first was a monolayer of
PAH, which uniformly coated the hydrophobic PDMS when deposited
under specific basic conditions (i.e., 50 mM, pH=10.5, 100 mM
NaCl). Next was the multilayered stack (in this case, containing
cytophilic hyaluronic acid) that became the bifunctional patch that
is attached to the cell. Next was a labile layer, e.g., prepared
from a low MW polyacrylamide and polyacrylic acid, a system known
to quickly degrade under neutral pH conditions (see, e.g., S. A.
Sukhishvili, S. Granick, Macromolecules 35, 301 (2002), which is
incorporated by reference in its entirety). Another structure for a
labile layer is a multilayer of poly(vinyl pyridine) and
poly(acrylic acid) (see, for example, J. Cho, F. Caruso,
Macromolecules 36, 2845 (2003), which is incorporated by reference
in its entirety). Using this same approach, Decher and co-workers
have shown the release of a free-standing PEM sheet using the
pH-dependant dissolution of a hydrogen-bonded sacrificial layer (S.
S. Ono, G. Decher, Nano Lett. 6, 592 (2006), which is incorporated
by reference in its entirety). The last stack was an adhesion layer
that that rendered the composite stack uniformly charged (either
cationic or anionic) at all pH conditions. This layer was then
brought into contact with the substrate, and the last layer (i.e.,
the adhesion layer) was chosen to be of opposite charge from the
stamping substrate to facilitate adhesion and lift-off.
[0080] A fluorescence microscope image of a composite multilayer
stack prepared by POPS
(PAH10.5).sub.1(PAA3.5/FITC-PAH7.5).sub.3(PAAm3/PAA3).sub.10(PAH4/SPS4).s-
ub.10 on a cationic glass slide is depicted in FIG. 4. The first
PEM deposited contained fluorescein (in the form of
fluorescein-labeled poly(allylamine hydrochloride) (FITC-PAH));
thus, the fluorescence observed indicated complete transfer of the
stack. The diameter of the posts was 5 .mu.m. A cytophilic stack
can have the structure
(PAH10.5).sub.1(HA7.5/PAH3.5).sub.x(PAAm3/PAA3).sub.10(PAH4/SPS4).sub.5,
where x indicates a variable number of bilayers.
[0081] A photolithographic lift off method was used to fabricate
3-ply polymer posts on a coated glass substrate The coating
included poly(diallyldimethylammonium chloride) (PDAC) and
poly(styrene sulfonate) (SPS), and had the structure
(PDAC4/SPS4).sub.15.5. The posts included PAA and
poly(4-vinylpyridine) had the formula
(PAA3/P4VP3).sub.10.5(CHI-FITC3/HA3).sub.3.5. Fluorescence images
of these posts are found in FIG. 5. High-fidelity posts were
obtained over very large areas (approximately 1 in.sup.2). The
ability of oppositely charged spheres to attach to these
cation-terminated posts was investigated, and a fluorescent image
showing these spheres is found in FIG. 6. The spheres are
carboxyl-functionalized polystyrene beads, 10.+-.0.56 .mu.m in
diameter, that were suspended in solution (pH approximately 5.5)
and incubated on top of the post-laden substrate shown in FIG. 5.
There are two intensities of fluorescent signal--more intense dots
are beads attached to the top of the post, and less intense dots
are attached to the substrate beneath. It was found that the beads
preferentially attached to the posts, despite the cation-terminated
PEM ((PDAC/SPS).sub.15.5) coating the substrate.
[0082] In a film that employed a diffusion barrier, exposure to PBS
allowed dissociation of the H-bonded releasable layer. FIGS. 7A-7B
are fluorescence micrographs of a field of 8 .mu.m posts of
(PAA3/PEG3).sub.10.5(PAH3/SPS3).sub.5.5(HA3/FITC-CHI3).sub.3.5,
shown (A) immediately after acetone lift-off, and (B) following 15
minutes of exposure to PBS. As can be seen in FIG. 7B, some
released patches rolled up or collapsed on themselves, whereas
others re-deposited flatly onto the cationic substrate. This
dissolution behavior was evidently limited to only approximately
10% of the total substrate area. Increasing the thickness of the
labile layer can encourage H-bonded release of fully-charged PEM
films built atop (PAA/PEG) (see, e.g., S. S. Ono, G. Decher, Nano
Lett. 6, 592 (2006), which is incorporated by reference in its
entirety).
[0083] Films using (PAA/PEG).sub.25.5 as the labile layer were
fabricated, and allowed more than 50% patch lift-off in 15 minutes
exposure to PBS at room temperature. FIGS. 8A-8B are fluorescence
micrographs of a field of 20 .mu.m posts consisting of a
(PAA/PEG).sub.25.5 labile layer, diffusion barrier, and a
FITC-labeled cytophilic layer, shown (A) immediately after acetone
lift-off, and (B) following 15 minutes of exposure to PBS. The use
of a diffusion layer can still be desirable, and the thickness of
the labile layer required for liftoff can depend on the presence of
a diffusion layer.
[0084] Interaction of polyelectrolyte multilayer-based patches with
CH27 murine B-lymphocytes was investigated. These cells were chosen
for their physiochemical similarity to e.g., human cytolytic T
lymphocytes, but are more robust than human T cells and have been
immortalized. B-lymphocytes are non-adherent cells that rarely
agglomerate, which makes the design of cell-patch interaction
schemes a unique challenge and opportunity.
[0085] Desirably, the interaction between the cell membrane and the
cytophilic layer does not substantially alter the cell's native
functions. Exemplary methods for encouraging the interaction of
cells with a cytophilic layer include the interaction between
lymphocyte cell-surface receptor CD44 and hyaluronic acid; the
non-selective decoration of a cell's surface with biotin and
streptavidin, paired with biotin on the surface of the patch; and
covalent crosslinking between reactive functional groups on the
patch and cell surface, either by direct reaction or by reaction of
each separately with functional groups on a crosslinking reagent.
More interaction mechanisms are possible, and are not limited to
the ones mentioned above.
[0086] A PEM patch was fabricated according to the following
formula: (PDAC4/SPS4).sub.15.5(PAA/PEG).sub.20.5(PAH3/SPS
3).sub.9.5(HA3/FITC-CHI).sub.3.5. A brightfield image of the
resulting B-cell array can be seen in FIG. 9. The diameter of each
cell is approximately 20 .mu.m.
[0087] Following the attachment of cells to the cytophilic layer of
a patch, the patches were released from the surface. The following
film composition was used:
(PAA/PEG).sub.25.5(FITC-PAH3/SPS3).sub.10(CHI3/HA3).sub.3. FIG. 10
shows an overlaid optical and fluorescent micrograph. The cells
marked by yellow arrows are freely floating in solution (i.e., have
been released from the surface), and were closely associated with
the fluorescein-labeled PEM patch (as indicated by the green
fluorescence).
[0088] Cell Adhesion by Stamping
[0089] A cytophilic, HA-terminated PEM was assembled on a PDMS
stamp. The PDMS stamp was then lowered onto a monolayer of CH27
cells anchored to hyaluronic acid spin-coated glass slides. The
cells then adhered to the HA-terminated PEM and delaminated a small
"plug" from the film. FIGS. 11A-11B show optical micrographs of
CH27 murine lymphocytes after 20 minutes of stamping with a
(PAH7.5/PAA3.5)10(FITC-CHI/HA)5 multilayer: (A) brightfield image
and (B) corresponding fluorescent image (due to the FITC-CHI
containing PEM).
[0090] Superparamagnetic Patches
[0091] Materials. Poly(methylacrylic acid) (PMAA, PolySciences,
M=100 kDa), poly(acrylic acid) (PAA, Aldrich, M=450 kDa),
poly(allylamine hydrochloride) (PAH, Aldrich, MW=70 kDa),
poly(ethylene glycol) (20 kMW-PEG, Aldrich, M=20 kDa),
poly(ethylene glycol) (1000 kMW-PEG, Aldrich, M=100 kDa),
poly(N-isopropylacrylamide) (PNIPAAm, Polymer Source, M=258 kDa),
fluorescein-labeled poly(allylamine hydrochloride) (FITC-PAH,
Aldrich, M=70 kDa), poly(diallyldimethylammonium chloride) (PDAC,
Aldrich, M=200-350 kDa in 20% aqueous solution), poly(styrene
sulfonate) (SPS, Aldrich, M=70 kDa), hyaluronic acid (HA, from
Strepococcus equi, Fluka, M approximately 145 kDa by intrinsic
viscosity (see, e.g., R. Mendichi, et al., Biomacromolecules 4 (6),
1805 (2003), which is incorporated by reference in its entirety)),
and low MW chitosan (CHI, DS=0.85, M approximately 390 kDa by
intrinsic viscosity) were used without purification. RMPI with
L-glutamine (Mediatech), Penicillin/Streptomycin (P/S, Mediatech),
fetal calf serum (characterized FCS, Mediatech), HEPES (VWR
Scientific), and NaHCO.sub.3 (VWR Scientific) were used for cell
culture media. Iron oxide magnetic nanoparticles
(Fe.sub.3O.sub.4NP, 10 nm diameter, Ferrotec EMG 705) stabilized
with an anionic surfactant were used. Fluorescein-labeled chitosan
was prepared according to the method of Tikhonov and Monfort (see
Vladimir E. Tikhonov, et al., Journal of Biochemical and
Biophysical Methods 60 (1), 29 (2004), which is incorporated by
reference in its entirety) and stored in a desiccator. Cells were
maintained and passaged in RPMI 1640 cell culture media
supplemented with 10% FCS, 5 mL/L P/S solution, 25 mM HEPES, and 18
mM NaHCO.sub.3.
[0092] Patterned multilayer heterostructure assemblies were
prepared by a traditional lift-off photolithographic approach to
pattern ultra-thin polymer films (see FIG. 2; see also J.
ShaikhMohammed, et al., Biomacromolecules 5 (5), 1745 (2004); and
J. ShaikhMohammed, et al., Langmuir 22 (6), 2738 (2006), each of
which is incorporated by reference in its entirety). A positive
photoresist was deposited onto a substrate, which is then exposed
with a 365 nm UV light source through a chromium-on-glass mask
patterned with 10 or 15 .mu.m diameter holes. After development,
the features in the photoresist extend down to the substrate. A
heterostructured polymer multilayer film was deposited conformally
on both the patterned photoresist and the exposed substrate. In the
final step, the sample was sonicated in acetone to dissolve the
photoresist and release the polymer film deposited on top. This
procedure left only the polymer that had been deposited within the
features and attached directly to the substrate. Using this
approach, uniformly patterned, heterostructured, surface-bound
patches could be made over areas as large as one square inch. This
fabrication method can be used to fabricate patches over larger
areas.
[0093] An aqueous-based layer-by-layer technique was used to
deposit the functional, heterostructured polymer film. This
technique has been used extensively for both polyelectrolyte and
hydrogen-bonded films (see, e.g., S. S. Shiratori and M. F. Rubner,
Macromolecules 33 (11), 4213 (2000); J. Choi and M. F. Rubner,
Macromolecules 38 (1), 116 (2005); and Gero Decher, Science 277
(5330), 1232 (1997), each of which is incorporated by reference in
its entirety).
[0094] Measurement of Patch Release Efficiency. Coordinate axes
were drawn on substrates containing surface-bound patch arrays,
which were photographed before exposure to neutral pH conditions.
The substrates were submerged in PBS and gently agitated on an
orbital shaker at 100 min.sup.-1. Using the coordinate axes,
identical regions were photographed before and after PBS exposure.
These before-and-after micrographs were compared, and each patch
was determined to have either not released (still on original
lattice site) or released and readsorbed onto the glass substrate.
The ratio of the number of non-released patches to the total number
of patches counted before exposure was recorded. Each value was the
average of at least three micrographs representing separate regions
on the substrate, which typically included approximately 300
patches.
[0095] Measurement of Cell-Patch Interaction Efficiency. To test
the binding efficiency of the cell-surface CD44 to
(HA/CHI)-terminated patches, such patches were fabricated without a
release region. B-cells in RPMI media (approximately 10.sup.6
cell/mL) were introduced on the surface, agitated on an orbital
shaker (100 min.sup.-1) for 15 minutes at room temperature, then
incubated at 37.degree. C. for 15 minutes. Some samples were
agitated and incubated a second time. At least 4 brightfield images
(surveying approximately 400 patches each) were analyzed for the
number of patches occupied with one cell, with multiple cells, the
number of empty patches, and the number of cells attached to
`interstitial` areas between patches.
[0096] Cell Functionalization. A patch-laden glass slide was cut
and placed in the bottom of a well in a 6-well plate. 2 mL of
B-lymphocytes suspended in RPMI media (approximately 10.sup.6
cells/mL) were pipetted onto the patch-laden surface. The entire
plate was agitated for 15 minutes at 37.degree. C., followed by
37.degree. C. incubation for 15 minutes. This agitation/incubation
cycle was repeated. Once on the surface of a patch, CD44 cell
surface receptors anchor onto the HA within the cell-adhesive
region. The glass slide, now containing lymphocytes attached to
surface-bound patches, was removed and gently shaken for
approximately 15 s upside down in 37.degree. C. PBS to remove all
cells not attached to a patch. The glass slide was returned to a
new well containing 4.degree. C. media, and the entire plate was
chilled to 4.degree. C. for 10 minutes. Confocal laser scanning
microscopy (CLSM) was used to image cells decorated with a
fluorescent polymer patch.
[0097] An adhesion layer, with formula (PDAC4/SPS4).sub.15.5 with a
typical thickness of 55 nm, made the substrate uniformly and
positively charged at all pH's and provides an adhesive surface for
subsequent depositions (see, e.g., Z. Z. Wu et al., Advanced
Materials 18 (20), 2699 (2006), which is incorporated by reference
in its entirety). This region was preferably deposited prior to
photoresist deposition and patterning, but it may also be deposited
onto the developed photoresist without affecting patch
fabrication.
[0098] The second region of the heterostructure was designed to
deconstruct (e.g., to release layers above from layers below)
readily upon exposure to physiological (or at least substantially
non-cytotoxic) conditions. Several deconstruction mechanisms may be
considered here, such as salt concentration, pH, or temperature
(see, for example, D. M. Lynn, Advanced Materials 19 (23), 4118
(2007), which is incorporated by reference in its entirety). A
hydrogen-bonded multilayer that deconstructs at physiological pH
but not at physiological temperature was chosen. PMAA/PNIPAAm films
dissolve above pH approximately 6.2, although other
hydrogen-bonding polymer combinations with higher critical pH
values may be used (see, e.g., Eugenia Kharlampieva and Svetlana A.
Sukhishvili, Polymer Reviews 46 (4), 377 (2006), which is
incorporated by reference in its entirety). The lower critical
solution temperature (LCST) behavior of PNIPAAm affords further
control over the release behavior--above the LCST temperature of
32.degree. C., polymer-polymer segment interactions are favored
over polymer-solvent interactions. In particular, polyanion/PNIPAAm
films demonstrate this characteristic through temperature dependent
diffusion behavior (see, for example, T. Serizawa, et al.,
Macromolecules 37 (17), 6531 (2004); and J. F. Quinn and F. Caruso,
Langmuir 20 (1), 20 (2004), each of which is incorporated by
reference in its entirety). The highly interdigitated nature of the
multilayered film does not allow the release region to dissolve
above the LCST. When the temperature drops well below the LCST,
(such as the commonly used 4.degree. C. condition used in tissue
culture work), the release region has both the pH and temperature
conditions needed to deconstruct and release the patch.
[0099] FIG. 20 illustrates the thermal release profile for a
PMAA/PNIPAAm release system. On the y-axis is the number of patches
remaining on the array after exposure to pH 7.4 PBS for 30 min at
the temperature shown on the x-axis. Very clear switching behavior
around the LCST of PNIPAAm homopolymer was observed, indicating
that the solubility of PNIPAAm primarily determines thermal release
behavior of the complexed PMAA/PNIPAAm multilayer.
[0100] When using PMAA/PNIPAAm, deposition of the labile layer, and
all following layers, must be done at a pH below 6.2 to prevent
premature film dissolution and release. Further, if PNIPAAm is used
for the release region, the deposition temperature must be below
32.degree. C. to ensure a true polymer solution. For several
hydrogen-bonded systems that can be used in the release region a
thickness of approximately 250 nm was desirable.
[0101] A payload multilayer assembly was deposited on top of the
labile layer. This layer of the patch is presented to the
extracellular environment after the patch is adhered to a cell and
released from the substrate on which it was assembled. Examples of
possible cargoes that may be incorporated into this region include
drugs, proteins, nanoparticles, environmentally responsive "smart"
materials, and imaging contrast agents. See, for example, M. C.
Berg, et al., Biomacromolecules 7 (1), 357 (2006); Y. Lvov, K.
Ariga, and T. Kunitake, Chemistry Letters (12), 2323 (1994); Y.
Lvov, et al., Thin Solid Films 285, 797 (1996); Z. Z. Wu, et al.,
Advanced Materials 18 (20), 2699 (2006); and Z. Li, et al.,
Langmuir 22 (24), 9820 (2006), each of which is incorporated by
reference in its entirety. Anionic, superparamagnetic nanoparticles
were alternately deposited with fluorescein-labeled PAH to create a
fluorescent labeled and magnetically responsive PEM patch. Ten
bilayers of magnetic nanoparticles and fluorescein-labeled PAH
yielded a 130 nm thick payload region.
[0102] The final region of the assembled heterostructure is the
cytophilic layer, anchoring the payload region to the cell
membrane. The cytophilic layer must be chosen with consideration of
the cells to be functionalized. For example, an (HA/CHI) multilayer
can be appropriate for functionalizing lymphocytes, because
lymphocytes contain CD44 cell-surface receptors whose natural
antigen is a 3-structural unit repeat of the polysaccharide
hyaluronic acid (see, e.g., C. Underhill, J Cell Sci 103 (2), 293
(1992), which is incorporated by reference in its entirety).
Previous work has shown how the molecular configurations of
adsorbed weak polyelectrolyte chains, including the relative
quantities of loops, trains, and tails, can be controlled using the
pH and salt conditions of the polymer solution. See, for example,
J. D. Mendelsohn, et al., Biomacromolecules 4 (1), 96 (2003); G. J.
Fleer, Polymers at interfaces, 1st ed. (Chapman & Hall, London
; New York, 1993, each of which is incorporated by reference in its
entirety. When in solution, a weak polyelectrolyte at a pH equal to
the solution pK.sub.a will have approximately half of the anionic
groups charged. The same polyelectrolyte, when incorporated into an
electrostatically crosslinked film, will have an "in-film" pKa that
is significantly shifted from the solution pKa. For an HA/CHI film
(HA pK.sub.a approximately 2.9) this effect can be used to maximize
the number of carboxylic acid-containing D-glucuronic acid sugar
units in HA that are uncharged and accessible to bind to CD44 by
adjusting the pH of the deposition solution. HA thus forms the
outermost layer of the cytophilic layer in each heterostructured
surface-bound patch. Chitosan was chosen as a complementary
polycation for its biocompatibility when complexed with HA in
multilayer films (see, e.g., A. L. Hillberg and M. Tabrizian,
Biomacromolecules 7 (10), 2742 (2006), which is incorporated by
reference in its entirety). Three and a half bilayers of HA and CHI
yields a approximately 20 nm thick cell-adhesive region.
[0103] An exemplary structure for a fluorescent, superparamagnetic,
lymphocyte-adhesive patch can be written as follows:
(PMAA3.0/PNIPAAm3.0).sub.x(FITC-PAH3.0/Fe.sub.3O.sub.4NP4.0).sub.y(CHI3.0-
/HA3.0).sub.z, with typical values x=80.5, y=10, and z=3.
[0104] The conditions that facilitate release of the patch from the
surface by the labile layer were investigated. Decher and Ono
previously reported that an electrostatic region built on top of a
hydrogren-bonded region requires a critical thickness of the
hydrogen-bonded region for successful dissolution and release (see,
e.g., S. S. Ono and G. Decher, Nano Lett. 6 (4), 592 (2006), which
is incorporated by reference in its entirety). The release
efficiency and release region thickness required for two very
different heterostructures, when agitated in PBS for 10 minutes at
room temperature, was determined. The two heterostructures
considered were as follows:
(PDAC4.0/SPS4.0).sub.15.5(PAA3.0/20kMW-PEG3.0).sub.x.5(PAH3.0/SPS3.0).sub-
.9.5(HA3.0/FITC-CHI3.0).sub.3.5 (see FIG. 12, and
(PMAA3.0/100kMW-PEG3.0).sub.x.5(FITC-PAH3.0/SPS3.0).sub.10 (see
FIG. 13). The release behavior for each heterostructure showed a
precipitous drop at a particular number of bilayers, above which
nearly all patches were released. The thickness required for
release, approximately 250 nm, was the same in all systems
examined, despite differences in polymers and molecular weight,
number of bilayers, and electrostatic PEM cap., the. The observed
critical thickness suggests that the polycation from the PEM cap
was diffusing into the hydrogen-bonded region to compensate the
surplus anionic charge found there. This polycation could
potentially electrostatically crosslink and stabilize the release
region. If the hydrogen-bonded region was thicker than the
diffusion path length of the polycation, some non-crosslinked
hydrogen-bonded chains remain to dissolve and release the patch
[0105] Controlling the kinetics of patch release can allow greater
flexibility in applications. For instance, some cell types may bind
more slowly than others, requiring more time to attach before
release; or reproduce very quickly, in which case release must be
very fast. A (PAA/PEG) labile layer releases rapidly, on the order
of a few seconds, and for many cell types, this is less time than
is required for sufficient binding between cell and patch. One
method for slowing down the release kinetics of a hydrogen-bonded
multilayer film is to increase the salt concentration of the
medium. Granick and co-workers have observed that some
hydrogen-bonded complexes are stabilized by increasing the ionic
strength of the solution (see, e.g., S. A. Sukhishvili and S.
Granick, Macromolecules 35 (1), 301 (2002), which is incorporated
by reference in its entirety). Charge-charge repulsion screening is
thought to be the origin of this effect, though this screening must
be stronger than the osmotic pressure created by the additional
associated ions, which favors film dissolution (see, for example,
Eugenia Kharlampieva and Svetlana A. Sukhishvili, Polymer Reviews
46 (4), 377 (2006), which is incorporated by reference in its
entirety). See the discussion above of the effect of salt
concentration displayed in FIG. 14.
[0106] The time required for patch release was also measured (see
FIG. 14). The following heterostructure was considered:
(PDAC4.0/SPS4.0).sub.15.5(PAA3.0/20kMW-PEG3.0).sub.20.5(FITC-PAH3.0/SPS3.-
0).sub.10(CHI3.0/HA3.0).sub.3. 80% of patches were released within
30 s when agitated in room temperature PBS, and virtually all
patches lifted off within 10 minutes. When 150 mM NaCl was added
(for a total of approximately 300 mM NaCl), it took 30 min to reach
a similar level of release. Error bars reflect the standard
deviation among 3 or more patch regions (approximately 300
patches).
[0107] Functionalization of B-Lymphocytes
[0108] Following acetone lift-off, a regular array of surface-bound
patches are observed on the glass substrate (see FIG. 15), each of
which is now free to anchor to a cell membrane via CD44-HA binding.
Preferably, cells settle to the patch-laden surface and associate
one cell per patch, though other outcomes are possible. First,
there may be more than one cell associated with a patch. Previous
studies on colloidal particles adsorbing on patterned surfaces have
shown that the ratio between the diameter of the particle and the
diameter of a circular feature will determine the cluster size
(see, for example, H. Zheng M. F. Rubner P. T. Hammond I. Lee,
Advanced Materials 14 (8), 572 (2002), which is incorporated by
reference in its entirety). During the lithography step, the
diameter of the patch can be easily controlled. It was found that
with 15 .mu.m diameter patches, many dimers (two cells per patch)
resulted, whereas with 10 .mu.m patches, monomers resulted almost
exclusively. Next, there are some cells that do not associate with
a patch, either because the cell remains in solution or settles
onto an `interstitial` area between patches. Adjusting the density
of surface patches and the number of cells in solution can control
this number. Additionally, patches may not attach to a cell but
will release in the neutral, 4.degree. C. media.
[0109] The relative frequency of these scenarios was investigated
by fabricating patches without a labile layer, and surveying the
cell attachment behavior to the patches and surface interstitial
areas. FIG. 16 shows that for the patch system
(Fe.sub.3O.sub.4NP4.0/FITC-PAH3.0).sub.10.5(CHI3.0/HA3.0).sub.3,
53.+-.5% of patches were occupied after 1 agitation/incubation
cycle, and that 85.+-.3% were occupied after 2 cycles (see FIGS.
17A-17B for representative optical micrographs of these two
scenarios). Previous attempts to immobilize and pattern
non-adherent CH27 B-cells required numerous steps involving polymer
stamping, antibodies, and non-selective attachment of biotin to the
cell surface (see, for example, H. Kim, et al., Advanced Functional
Materials 16 (10), 1313 (2006); and H. Kim, et al.,
Biomacromolecules 5 (3), 822 (2004) each of which is incorporated
by reference in its entirety). Here, a straightforward method for
non-adherent cell patterning based solely upon the natural
interaction between CD44 and HA is provided, with an efficacy that
rivals the previous method.
[0110] An important design parameter is the balance between cell
adhesion and dissolution of the labile layer. The strength of the
interaction between the cell and the cell-adhesive layer is
desirably greater than that between the functional and release
regions. If the patch lifts off the substrate before a cell is able
to bind, the likelihood of the patch encountering a cell (while
both are floating freely in dilute solution) is very low. Other
options for cell-patch interaction exist, such as non-selective
biotin/streptavidin or RGD-integrin strategies. See, for example,
H. Kim, et al., Advanced Functional Materials 16 (10), 1313 (2006);
H. Kim, et al., Biomacromolecules 5 (3), 822 (2004); and M. C.
Berg, et al., Langmuir 20 (4), 1362 (2004), each of which is
incorporated by reference in its entirety. Temperature also plays a
role in mediating the cell-patch and labile layer dissolution.
Lymphocytes did not attach to HA-containing surfaces at 4.degree.
C., and most efficiently bound at 37.degree. C. Using an LCST-based
labile layer allows us to attach the cells at a temperature optimal
for encouraging attachment and preventing release. The temperature
can then be very briefly lowered below the LCST to encourage patch
release, and then the cells returned to physiological
temperature.
[0111] FIG. 18 shows CLSM optical brightfield and fluorescence
images of a patch on the surface of two live lymphocytes suspended
in media immediately after patch attachment. FIG. 18a shows a patch
attached to the surface of a CH27 B-lymphocyte, and FIG. 18b is a
HuT78 T-lymphocyte.
[0112] To test that magnetic properties were conferred on the cell
via the attached patch, B-lymphocytes were exposed to
superparamagnetic patches containing a PMAA/PVPON-release region.
The free-floating lympocytes were imaged in a LabTek chamber using
an inverted microscope. After cells were allowed to settle, a rare
earth magnet was placed close to the imaged region but outside of
the chamber. FIG. 19 shows how a CH27 cells responds to the applied
magnetic field as a result of the membrane-bound patch. This cell
moved a total of approximately 200 .mu.m in 11 seconds, but moves
out of the focal plane during the course of imaging.
[0113] Using commercially available amino-functionalized quantum
dots (600 nm emission), the patches were prepared according to the
following:
(PMAA3/PNIPAAm3).sub.x.5(QuantumDots5/SPS5).sub.30(PAH4/MNP4).sub.10(CHI3-
/HA3).sub.3 where x is 20, 40, 60 or 80 (MNP=magnetic
nanoparticles). Neutral pH conditions resulted in successful
release. FIGS. 21A-21C, 22A-22C, and 23A-23C are micrographs of the
patches in the absence (FIG. 21) or presence (FIGS. 22 and 23C) of
CH27 B-cells. In these figures, panel A is a fluorescence image,
panel B is a bright field micrograph of the same subject, and panel
C is a merged image of panels A and B.
[0114] The hyaluronic acid-CD44 interaction was effective for
associating patches with cells (e.g., B cells) displaying CD44 on
the cell surface. However, not all cell types display CD44, and so
a more general mechanism of association can be desirable. Nearly
all cell types bear free thiol (--SH) groups on the cell surface.
Thiol groups can react with other functional groups (such as, e.g.,
a maleimide group) and this reactivity can be used to form a
covalent bond between a patch and the cell surface.
[0115] In particular, a patch may be exposed to a crosslinking
reagent prior to be exposed to cells. The crosslinking reagent can
be chosen to have two reactive groups with differing reactivity, so
that one reactive group can form a covalent bond with a functional
group found in the patch, and the other reactive group can form a
covalent bond with a thiol group. For example, the crosslinking
reagent can include an amine-reactive functional group (such as,
for example, an N-hydroxysuccinimide group, capable of reacting
with amine groups in, for example, PAH) and a thiol reactive group.
Other suitable reactive groups and crosslinking reagents are
known.
[0116] Patches were exposed to heterobifunctional crosslinking
reagents, and subsequently to a free-thiol containing fluorescent
dye. The absorbance of this dye was measured for different
heterobifunctional crosslinkers, and it was found found that the
maleimide group was covalently attached to the patch. FIG. 24 plots
absorbance intensity of patches after crosslinking to the dye,
showing that both heterobifunctional crosslinkers successfully
presented thiol-reactive maleimide groups. The crosslinking
regeants used were
N-[.kappa.-maleimidoundecanoyloxy]sulfosuccinimide ester
(sulfo-KMUS) and
succinimidyl-[(N-maleimidopropionamido)-octaethyleneglycol] ester
(SM(PEG).sub.8), both available from Pierce Biotechnology. Other
crosslinking reagents are commercially available, from Pierce and
other suppliers.
[0117] A covalent linking approach was used to associate patches
with dendritic cells, another type of immune cell. Dendritic cells
belong to a large class of cells called antigen-presenting cells,
or APCs, which are known to be phagocytotic. In general, these
cells will internalize and digest anything found in their
environment, presenting fragments of resulting antigens on their
surface, in turn stimulating further immune system responses. FIGS.
25A-25C are micrographs of a (PMAA3/PNIPAAm3).sub.80.5
(PAH4/MNP4).sub.10(CHI3/HA3).sub.3 patch associated with a
dendritic cell spread on a tissue culture polystyrene (TCPS) dish
(panel A, a fluorescence image; panel B bright field microscopic
image of the same subject; and panel C, merged image of panels A
and B). FIGS. 26A-26C are micrographs of a
(PMAA3/PNIPAAm3).sub.80.5(PAH4/MNP4).sub.10patch associated with a
dendritic cell on a PDAC-terminated background.
[0118] Unlike nano- or microparticle based delivery systems, these
phagocytes did not internalize the patches. Cells were observed
with patches attached, then being released from the cell surface,
and then being picked up again. Cells with patches attached were
also observed to phagocytose small polystyrene beads, demonstrating
that the cell was still able to perform its native function
(phagocytosis) without interference from the patch. It also
demonstrated that the patch can remain exposed to the extracellular
environment and able to deliver drug, adjuvants, or antigens to the
local surroundings while the cell remains able to phagocytose and
process antigens for presentation.
[0119] Association of a patch with an immortalized human T-cell did
not impair the cell's native migration capabilities. After
association, the cell was allowed to migrate on a protein-coated
coverslip and observed microscopically. The cell was still able to
migrate, and always kept the patch on the trailing end of the cell.
This cell was seen to migrate for several hours with the pactch
attached. Images of this T cell recorded at three different time
points are found in FIG. 27.
[0120] Other embodiments are within the scope of the following
claims.
* * * * *